Practical Accomplishment of
the validation for Blood
Groups Genotyping in KCBB
Hana Al-Shatti
Head of Molecular Laboratory
Kuwait central blood Bank
Introduction
The Validation of reagents used for blood typing is
a very well known issue in blood transfusion
practice, it is usually done by following universal
blood banks standards such as AABB standard
Validating reagents for heamagglutination
reactions is easier than that of molecular
genotyping, as the later application interferes
with machines, specific equipments as well as
a very low volumes reagents.
(ranges: 0.5µl – 100 µl)
Test Description
Bloodchip detects the genotype by analyzing fluorescence
signals corresponding to hybridization rate between labeled
amplified DNA fragments and probes attached to the surface of
the chip glass.
Blood chip test analyzes more than 130 polymorphisms
determining the blood groups: ABO, Rh, Kell, Kidd, Duffy, MNS
, Diego, Dombrock , Colton, Lutheran and HPA.
Work Flow
The DNA Sample is amplified in three different PCR
reactions, After which it will be fragmented and labeled with
two different flurophores.
The labeled PCR products are applied to the surface of the
microarray slide for hybridization.
Finally the slides are scanned by microarray scanner.
Work Flow
1
Amplification
PCR mix 1, 2 , 3
5ul DNA
(20ng/ul)
3hours 30 minutes
2
Fragmentation
DNAse
+
Alkaline Phosphatase
3
Labelling
4
5
Hybridization Results analysis
TdT+ BiotinddUTP or dCTPCy5
10
ul
45 minutes
60 minutes
3 hours 30 minutes
The Data analysis software generates the genotype results as
well as the phenotype for each allelic variants of all blood
groups Included in the software.
The blood chip platform was installed by the
supplier engineers and consisted of .
(A)Pre PCR area
1- DNA extraction machine.
2- spectrophotometer.
3- PCR mix preparations.(PCR
cabinet)
(B) Post PCR area
1- Thermal cycler.
2- hybridization station.
3- slides Scanner.
After installation, all installation qualification
documents were signed by the project leader.
what was done to validate Bloodchip??
Validation: the whole process of establishing documented
evidence which provides a high degree of assurance that
the performance of process (including equipments) will
consistently produce a product that meet its pre-determined
specifications and quality attributes.
Validation procedure:1) In Kuwait central Blood bank, we have started our
validation process by forming the blood chip validation
committee; a group of field experts, quality assurance
staff, operational users, IT staff, engineering staff and
the project leader.
This committee was approved by our medical director.
2) Operator's training was completed for technologists by
the manufacturer’s experts in a period of 4 weeks in
molecular lab of Kuwait central blood bank as well as one
week in the manufacturer’s facility in Spain.
3)The Designing of validation flow chart related to
samples and genotyping results; a flow chart that shows
the kind of samples to be tested during validation and the
kind of tests to be performed .
The samples were chosen to cover donors and patients.
Bloodchip validation Flow chart
Internal validation Results :
(1)genotyping and serological phenotyping results were
100% matching , ( except for some variants that were
not comparable as these are not available as
serological reagents) .
(2) the HPA genotyping results were also a 100 %
matching when compared with the serology using
pack 12 and HPA1 typing assays.
(3)The test is consistent; the 4 different technologists
performed the blood chip validation testing have had an
identical results for all the samples.
Externally:
(1) we have participated in an external exercise with NIBSC
(National Institute for Biological Standards and Control) in the
United kingdom. This exercise is specially for human platelets
antigen (HPA) typing.
During our validation period we have participated in 3 exercises
(exercise# 2010-B, 2011-A, 2011-B) each included 4 sample to
be genotyped for HPA-1 ,-2,-3,-4,-5,-6 and -15.
Results were very pleasant as we had a 100% correct
genotyping.
(2) The samples for two patients were sent to the
platelet immunology reference lab in Cambridge, UK for
HPA -1,-2,-3,-4,-15 for result confirmation. The results
were completely matching to our Blood chip results.
(3) 15 samples were sent to blood chip manufacturer
facility ( progenika) in Spain to be tested in parallel to
Kuwait blood chip testing, and the results were 100 %
identical.
The validation committee agreed that the test bloodchip is
valid to be used as the blood groups genotyping test in
Kuwait central blood bank and that was approved by our
medical director .
Performance Qualification
Definition
The last of the qualification processes and should only
commence after successful completion of operational
qualification .The output of the process being validated
should demonstrate satisfactory performance over the full
range of expected operating conditions.
During 6 months testing following the validation of the
bloodchip, (420 tests)
1) Contamination problem (once ).
2) Not valid results (14%).
3) No call results (9%).
Conclusion:
Validation process of Blood chip was not an easy task to do, we
have actually succeeded in recruiting experts in the field, an
advanced genotyping technology and a lot of specific
requirements to Validate such an accurate testing. We were
also able to overcome difficulties in the process of results
confirmation and comparison with other institutes abroad for
the purpose of full filling the validation requirements which
satisfies our aspirations in:
1) Pushing up advancements of blood grouping in
KCBB.
2) Continuing our mission of earning more
accreditations.
Special thanks to my colleagues in the validation
committee :
1) Dr.Reem Al-Radwan (Medical director of KCBB)
2) Ohood Al-Ayyadi
(Lab department manager)
3) Alia Al-failakawi
(Molecular lab staff)
4)Ohood Al-shaikh
(Molecular lab staff)
5) Eng.Suleiman Qasem(ATC)
6) Dr.Rachid Al-hems
(Progenika)
Hana Al-Shatti